The pulsatile secretory activity of the GnRH-producing neuronal network in the hypothalamus, and consequently of pituitary gonadotroph cells, is essential for the maintenance of normal patterns of gonadotropin secretion and reproductive function. Based on the capacity of cultured fetal hypothalamic cells and immortalized GnRH neurons (GT1-7 cells) for prolonged pulsatile release of GnRH, we have analyzed the cellular and biochemical mechanisms of episodic neurosecretion in vitro. Pulsatile GnRH secretion is highly calcium-dependent, and is stimulated by increased cell excitability and cyclic AMP (cAMP). The GnRH secretory profile is also influenced by agonist activation of the endogenous GnRH receptor (GnRH-R), which couples to Gq/11 as indicated by reduction of membrane-bound Galpha-q/11 and increased inositol phosphate/Ca2+ signaling. In contrast, GnRH antagonists abolish pulsatile GnRH secretion and increase membrane-associated alpha-q/11. GnRH stimulates cAMP production at low nanomolar concentrations but at high concentrations has an inhibitory effect that is abolished by pertussis toxin (PTX). Coupling of the GnRH-R to both Gs and Gi proteins was also indicated by the ability of nanomolar GnRH concentrations to reduce membrane-associated alpha-s and alpha-i3 levels, and of high concentrations to diminish alpha-i3 levels. Conversely, alpha-i3 was increased during GnRH antagonist and PTX treatment, with concomitant loss of pulsatile GnRH secretion. In cholera toxin (CTX)-treated GnRH neurons, decreases in alpha-s immunoreactivity and increases in cAMP production paralleled the responses to nanomolar GnRH concentrations. It is noteworthy that treatment with CTX and 8-Bromo-cAMP amplify episodic GnRH pulses but do not affect their frequency. These findings indicate that an agonist concentration-dependent switch in coupling of the GnRH-R between specific G proteins regulates Gq/11-InsP3/Ca2+ signaling, as well as Gs-cAMP stimulatory and Gi-cAMP inhibitory reponses. A model has been proposed in which this autocrine inhibitory mechanism serves as a timer to regulate the frequency of Ca2+- and cAMP-dependent episodes of GnRH release. In current studies, the mechanism by which GnRH activates ERK MAP kinase in GT1-7 neurons, and the role of transactivation of the EGF receptor therein, is under investigation. We have observed that hypothalamic GnRH neurons and their immortalized counterparts (GT1-7 cells) express not only nuclear but also cell membrane receptors for estrogen receptors (ERalpha and ERbeta). Both cell types exhibit positive immunostaining for plasma-membrane ERs, as well as estradiol-induced changes in adenylyl cyclase activity. In GT1-7 cells, physiological (picomolar) estradiol concentrations cause dose-dependent inhibition of cAMP production that is abolished by the ER antagonist, ICI 182,780. Estradiol-induced inhibition of adenylyl cyclase in cells and membranes is also prevented by treatment with PTX, consistent with coupling of the membrane-bound estradiol receptors to an inhibitory G protein. Recent studies have indicated that the negative regulatory action of estradiol on cAMP production is associated with a direct interaction between ERalpha and the Gi alpha-subunit, as demonstrated by immunoprecipitation with a specific anti-ERalpha antibody. In perifused GT1-7 cells and hypothalamic neurons, treatment with ovulatory phase estradiol levels prolongs the GnRH inter-peak interval, shortens peak duration, and increases peak amplitude. These findings have demonstrated that the membrane-associated ER expressed in GnRH neurons exhibit high-affinity interactions with adenylyl cyclase inhibitory G proteins by a rapid non-genomic mechanism, and modulate intracellular cAMP signaling and neuropeptide secretion. The high agonist sensitivity of this interaction is commensurate with the low estradiol concentrations at which Gi-mediated inhibition of cAMP production is observed, and suggests that this process represents a physiological negative feedback action of estrogen on the GnRH neuron. The nature of the membrane-associated estradiol receptor, and its relationship to the endogenous nuclear receptor of the GnRH neuron, are currently under investigation.